Composition and methods using myelin-associated glycoprotein (MAG) and inhibitors thereof

ABSTRACT

This invention relates to the novel identification of myelin-associated glycoprotein (&#34;MAG&#34;) as a potent inhibitor of neural regeneration. More particularly, this invention relates to compositions and methods useful for reversing inhibition of neural regeneration in the central and peripheral nervous system. Assays to monitor the effects of MAG on neural regeneration and to identify agents which will block or promote the inhibitory effects of MAG on neural outgrowth are provided. Screening methods for identifying such agents are also provided. This invention also relates to compositions and methods using agents that can reverse the inhibitory effects of MAG on neural regeneration. Methods for regulating and for promoting neural growth or regeneration in the nervous system, methods for treating injuries or damage to nervous tissue or neurons, and methods for treating neural degeneration associated with disorders or diseases, comprising the step of administering at least one of the compositions according to this invention are provided.

Benefit of United States Provisional Application No. 60/000,561, filedJun. 27, 1995, is claimed herefor.

TECHNICAL FIELD OF THE INVENTION

This invention relates to the novel identification of myelin-associatedglycoprotein ("MAG") as a potent inhibitor of neural regeneration. Moreparticularly, this invention relates to compositions and methods usefulfor reversing inhibition of neural regeneration in the central andperipheral nervous system. Assays to monitor the effects of MAG onneural regeneration and to identify agents which will block or promotethe inhibitory effects of MAG on neural outgrowth are provided.Screening methods for identifying such agents are also provided. Thisinvention also relates to compositions and methods using agents that canreverse the inhibitory effects of MAG on neural regeneration. Methodsfor regulating and for promoting neural growth or regeneration in thenervous system, methods for treating injuries or damage to nervoustissue or neurons, and methods for treating neural degenerationassociated with disorders or diseases, comprising the step ofadministering at least one of the compositions according to thisinvention are provided.

BACKGROUND OF THE INVENTION

The mammalian nervous system does not regenerate after injury despitethe fact that there are many molecules present which encourage/promoteaxonal (nerve) growth. It is believed that the lack of regenerationcaused by the presence of molecules in the central nervous system (CNS)and the peripheral nervous system (PNS) which actively prevent/inhibitregeneration. Hence, the well documented inability of the adultmammalian CNS to regenerate after injury is believed to result from apredominance of inhibitory molecules.

It has been demonstrated that when neurons are grown on tissue sectionsof the CNS they fail to extend processes onto areas of white matter,myelin. It is believed that myelin-specific inhibitory molecules canlargely account for the lack of CNS regeneration and theiridentification will help in the design of therapies to encourageregrowth after injury. The precise molecules responsible for thisinhibition have, so far, remained elusive. If these inhibitory moleculescan be identified and blocked, then neural regeneration can beencouraged.

Schwab and co-workers have identified two components in CNS myelin, inthe molecular weight ranges of approximately 35 kD and 250 kD, whicharrest axonal growth. The most compelling observation in support of theinhibitory action of these two protein fractions is that antibodiesraised to proteins eluted from these regions of polyacrylamide gelsafter separation of CNS myelin proteins, specifically reverses theinhibitory effect of myelin in vitro and allows limited spinal cordregeneration when applied in vivo to transected nerves (Caroni, P. andSchwab, M. E., Neuron, 1, pp. 85-96 (1988a); J. Cell Biol., 106, pp.1281-88 (1988b); Schnell, L. and Schwab, M. E., Nature, 343, pp. 269-72(1990)). The nature of these two proteins and how they act have not yetbeen described, but, it is generally accepted that they are significantcontributors to the inhibitory effect of this tissue. However, asacknowledged by the authors, other factors are likely to contribute tothe inhibition by CNS myelin as even in the presence of antibodiesdirected against these two proteins, the majority of axons in vivo failto regenerate (Schnell, L. and Schwab, M. E., Nature, 343, pp. 269-72(1990); Schnell et al., Nature, 367, pp. 170-73 (1993)).

In addition to inhibitory molecules in myelin, another family ofproteins has recently been identified whose members inhibit axonalregeneration. These molecules are called collapsins (Luo et al., Cell,75, pp. 217-27 (1993)). However, collapsins are found ubiquitouslythroughout the nervous system and as they are found in regions of thenervous system in which axons will grow, i.e. gray matter, they areunlikely to contribute significantly to the lack of neural regenerationafter injury. Instead, the collapsins most likely play a role in guidinggrowing axons during development.

Previously it was shown that MAG, like many members of theIg-superfamily of molecules, could promote neurite outgrowth, in thiscase, from dorsal root ganglion (DRG) neurons from 2 day old rats(Johnson et al., Neuron, 3, pp. 377-85 (1989)). We observed a similareffect on DRG neurons from rats up to postnatal day 3, but after thisage MAG had the opposite effect, i.e., it inhibited neurite outgrowth(Mukhopadhyay et al., Neuron, 13, pp. 757-67 (1994)). Furthermore, wealso found that MAG dramatically inhibited neurite outgrowth fromcerebellar neurons from rats of all ages up to adult. Polyclonalantibodies directed against MAG could specifically block bothstimulatory and inhibitory effects of MAG on neurite outgrowth. MAG,therefore, depending on the age and the type of neuron, can eitherpromote or inhibit neurite outgrowth. Subsequent to our report on theinhibitory effects of MAG, another group demonstrated, using a differentcomplementary approach, that MAG is an inhibitor of axonal growth(McKerracher et al., Neuron, 13, pp. 805-811 (1994); WO 95/22344 (Aug.24, 1995); incorporated herein by reference).

It would be useful to block the inhibitors of axonal regeneration fortreating patients with nervous system injuries where neural regenerationis a problem. No molecule had been identified in myelin which is apotent inhibitor of axonal regeneration. Although Schwab and co-workersidentified components in myelin that are inhibitory, the precise natureof these components has not been identified, i.e., they have not beencloned nor have the proteins been purified. In addition, there was noinformation available on the component on the neuron that the putativeinhibitory molecules interact with to prevent regrowth. As no inhibitorynor interacting molecules had been precisely identified, it wasdifficult, if not impossible, to logically design strategies wherebythese molecules can been blocked and prevented from inhibiting neuralregeneration.

SUMMARY OF THE INVENTION

The present invention solves the problems referred to above byidentifying MAG as a potent inhibitor of axonal regeneration in thecentral nervous system (CNS) and the peripheral nervous system (PNS).The present invention provides compositions and methods for blocking ormanipulating the levels of MAG activity in the nervous system.

In one embodiment, the compositions comprise a pharmaceuticallyacceptable carrier and a therapeutically effective amount of at leastone inhibitor of MAG. Inhibitors of MAG include but are not limited toanti-MAG antibodies, altered and/or mutated forms of MAG characterizedby an altered biological activity, free sialic acid-bearing sugars,modified derivatives of sialic acid attached to a sugar, a sialicacid-bearing sugar attached to a protein or lipid carrier molecule, amodified sialic acid-bearing sugar attached to a protein or lipidcarrier molecule and a sialic acid glycopeptide.

In one preferred embodiment, the MAG inhibitor comprises a small sialicacid-bearing oligosaccharide (sugar), which is optionally a competitiveinhibitor of sialidase. More preferably, the sialic acid analog is sialo2,3-α lactose (2,3-SL) or 2,3-dideoxy sialic acid (DD-NANA).

In another preferred embodiment, the MAG inhibitor comprises an alteredand/or mutant form of MAG which can inhibit the binding of endogenousMAG to neurons in the CNS or PNS. Altered forms of MAG preferablycomprise all or a portion of the extracellular domain of MAG fused toanother molecule which renders the chimeric protein soluble. One suchpreferred soluble MAG chimeric protein comprises the five Ig-likedomains of MAG fused to the Fc domain of a human immunoglobulinmolecule, such as IgG ("MAG-Fc").

Preferred altered/mutated forms of MAG are soluble molecules whichharbor one or more mutations in the MAG molecule that reduce oreliminate its ability to inhibit or promote neurite outgrowth comparedto endogenous MAG or MAG-Fc, but do not significantly diminish thebinding of the altered or mutant form of MAG to neuronal surfaces. Mostpreferred altered/mutant forms of MAG are soluble molecules comprising atruncated form of MAG-Fc consisting of the first three of the fiveextracellular Ig-like domains of MAG fused to an immunoglobulin Fcdomain ("MAG(d1-3)-Fc").

In another embodiment, the compositions comprise a therapeuticallyeffective amount of an enzyme that can alter or remove sialic acidresidues having a Neu5Acα2→3Galβ1→3GalNAc (3-O) structure, which mediateMAG binding to neuronal surfaces in the PNS or CNS. Preferredcompositions of this embodiment comprise sialidase (a neuraminidase) andsialyl transferases that alter the structure and/or lower the effectiveconcentration of Neu5Acα2→3Galβ1→3GalNAc ("3-O") sialyated glycans.

The present invention also provides methods for regulating and forpromoting neural growth or regeneration in the nervous system, methodsfor treating injuries or damage to nervous tissue or neurons, andmethods for treating neural degeneration associated with disorders ordiseases, comprising the step of administering at least one of thepharmaceutical compositions according to this invention.

The present invention provides an assay for determining whether neuriteoutgrowth from a particular type of neuron at a particular age isstimulated or inhibited in the presence of MAG (or a MAG derivative). Inone embodiment, the method comprises the steps of:

a) culturing a first sample of a selected neuronal cell type on agrowth-permissive substrate in the absence of MAG;

b) culturing a second sample of the selected neuronal cell type on agrowth-permissive substrate comprising bound MAG; and

c) comparing the relative amount of neurite growth in the cultured cellsof a) and b); wherein when the relative growth of neurites in thecultured cells of a) is greater than in b), the neuronal cell isinhibited by the presence of MAG, and when the relative growth ofneurites in the cultured cells of a) is less than in b), the neuronalcell is stimulated by the presence of MAG.

In a preferred embodiment, the growth-permissive substrate in theabsence of MAG comprises a monolayer of mammalian cells that do notexpress cell-surface MAG, and the growth-permissive substrate comprisingbound MAG comprises a monolayer equivalent mammalian cells engineered toexpress cell surface MAG. Preferably, the mammalian cells are CHO cellsengineered to express cell surface MAG, such as CHO-MAG2 cells.

The present invention also provides methods for identifying aMAG-dependent neurite growth altering agent, i.e., an agent which altersneurite outgrowth from a selected neuronal cell type, or population ofmixed cell types, in the presence of MAG compared to the absence of MAG.

In one embodiment, the method comprises the steps of:

a) culturing a first sample of a selected neuronal cell type on agrowth-permissive substrate in the absence of MAG;

b) culturing a second sample of the selected neuronal cell type on agrowth-permissive substrate comprising bound MAG in an amount sufficientto alter neurite outgrowth from the cells compared to the first sampleof cells cultured in the absence of MAG;

c) incubating the cell cultures of a) and b) with a known relativeconcentration of a test agent for a time sufficient to allow neuritegrowth; and

d) comparing the relative amount of neurite growth in the cultured cellsof a) and b); wherein an agent that changes the relative growth ofneurites in the cultured cells of a) and b) is identified as aMAG-dependent neurite growth altering agent.

In a preferred embodiment, the growth-permissive substrate in theabsence of MAG comprises a monolayer of mammalian cells that do notexpress cell-surface MAG, and the growth-permissive substrate comprisingbound MAG comprises a monolayer equivalent mammalian cells engineered toexpress cell surface MAG. Preferably, the mammalian cells are CHO cellsengineered to express cell surface MAG, such as CHO-MAG2 cells.

In another embodiment of this invention, the method for identifying aMAG-dependent neurite growth altering agent comprises the steps of:

a) culturing separate samples of a selected neuronal cell type on agrowth-permissive substrate lacking MAG;

b) culturing a first sample of a) with a known concentration of atraceable, soluble form of MAG;

c) culturing a second sample of a) with a known concentration of atraceable, soluble form of a control protein lacking MAG activity;

d) incubating the cultures of b) and c) with a known relativeconcentration of a test agent for a time sufficient to allow neuritegrowth; and e) comparing the relative amount of neurite growth in thecultured cells of c) and d); wherein an agent that changes the relativegrowth of neurites in the cultured cells of c) and d) is identified as aMAG-dependent neurite growth altering agent.

In one preferred embodiment, the growth-permissive substrate lacking MAGcomprises a monolayer of mammalian cells that do not expresscell-surface MAG, such as COS or NIH 3T3 cells. In another preferredembodiment, the growth-permissive substrate lacking MAG comprises animmobilized monolayer of a purified, growth-promoting factor. One mostpreferred neuronal growth-promoting factor which may be immobilized ontoa monolayer is the L1 glycoprotein.

In preferred embodiments, the soluble form of MAG is a MAG-Fc fusionprotein, and the soluble control protein lacking MAG activity is a MUC18-Fc fusion protein. Preferred traceable fusion proteins areradioactively or fluorescently labeled.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Inhibition of Neurite outgrowth from Cerebellar Neurons by MAG.Cerebellar neurons from post-natal day (PND) 1, 4 and 7 were grownovernight on a monolayer of MAG-expressing (clear bars) or controltransfected CHO cells (hatched bars). Neurons were stained for GAP-43antigen and neurite length was measured and the average lengthcalculated from at least 150 measurements (±SEM).

FIG. 2. Effect of NAG on Neurite Outgrowth from DRG Neurons of DifferentAges. DRG neurons were isolated from animals from PND 1 to PND 20 andgrown on MAG-expressing or control CHO cells and neurite length wascalculated as described in FIG. 1.

FIG. 3. Effect of MAG on Neurite Outgrowth from Different Neuronal CellTypes. Various neurons from PND1 animals were isolated and grownovernight on MAG-expressing (dark hatched) or control (light hatched)CHO cells. Average neurite length was calculated as described in FIG. 1.RG=retinal ganglion; HN=hippocampal; MN=motor; and SCG=superior cervicalganglion neurons.

FIG. 4. Sialic acid-dependent Binding of MAG to Cerebellar and DRGNeurons. Radiolabelled MAG-Fc (solid bars) was allowed to bind to PND 1cerebellar and DRG neurons. Incubations were also carried out in thepresence of 5 μg/ml of MAG 513 monoclonal antibody (dark hatched bars)or with neurons that had been desialyated (speckled bars). Eachexperiment was carried out in quadruplicate. The results are the mean of3 experiments.

FIG. 5. Desialation of Neurons Blocks the Inhibition of NeuriteOutgrowth by MAG. Neurite outgrowth was compared, as described in FIG.1, for cerebellar neurons from PND 2 animals on MAG-expressing andcontrol CHO cells, with and without desialation of the neurons beforethe assay. C=neurons grown on control CHO cells; MAG=neurons grown onMAG-expressing CHO cells; +=neurons were desialyated prior to the assay;++=neurons were desialyated prior to the assay and desialidase wasincluded in the cultures. Results represent the average neurite length(μm) from at least 150 neurons±SEM.

FIG. 6. Desialation of Neurons Blocks the Promotion of Neurite outgrowthby MAG. Neurite outgrowth was compared, as described in FIG. 1, for DRGneurons from PND 1 animals on MAG-expressing (MAG) and control CHO cells(Control), with (MAG sialidase) and without desialyation of the neuronsbefore the assay. Results represent the average neurite length (μm) fromat least 150 neurons±SEM.

FIG. 7(a). Soluble MAG-Fc Inhibits Axonal Regeneration of CerebellarNeurons Grown on L1 in a Concentration-dependent Manner. L1-Fc wasimmobilized and isolated PND2 cerebellar neurons were grown overnight inthe presence of various concentrations of MAG-Fc (diamonds) or MUC-Fc(squares) as indicated. Neurons were fixed, stained and neurite lengthmeasured as described in FIG. 1. Results represent the average neuritelength (μm)±SEM.

FIG. 7(b). MAG-Fc Inhibits Axonal Growth in a Specific, SialicAcid-dependent Manner. Cerebellar neurons were grown on immobilized L1as a substrate. MAG-Fc (column 1) or MUC-Fc (column 2) were added at aconcentration of 50 μg/ml. Anti-MAG 513 monoclonal antibodies wereincluded at a concentration of 5 μg/ml (column 3) or desialyated neuronswere used (column 4). Neurite length (μm) was measured as described inFIG. 1. Results represent the average neurite length (μm)±SEM.

FIG. 7(c). MAG-Fc Inhibits Axonal Growth from Neurons Grown onFibroblasts. Isolated PND 2 cerebellar neurons were grown on a substrateof fibroblasts (3T3 cells) in the presence of 50 μg/ml MAG-Fc (Column 1,MAG), 50 μg/ml of MUC18-Fc (column 2, MUC 18) or in the presence of 5μg/ml anti-MAG 513 monoclonal antibodies (column 3, anti-MAG). Neuritelength (μm) was measured as described in FIG. 1. Results represent theaverage neurite length (μm)±SEM.

FIG. 8(a). MAG(d1-3)-Fc Binds to Neurons in a Specific, SialicAcid-dependent Manner. Cerebellar (PND2) neurons vitally labeled withfluorescein were allowed to bind to immobilized MAG(d1-5)-Fc (darkbars), MAG(d1-3)-Fc (hatched bars) or MUC18-Fc (speckled bars), in thepresence (+antiMAG) or absence (-Ab) of an anti-MAG monoclonal antibody.Results represent the amount of Fc-chimera bound (ng).

FIG. 8(b). MAG(d1-3)-Fc Does Not Inhibit Axonal Regeneration. Cerebellarneurons were grown on immobilized L1-Fc, in the presence of 50 μg/mlMAG(d1-5)-Fc, Muc18-Fc, or MAG(d1-3)-Fc. Neurite length (μm) wasmeasured as described in FIG. 1. Results represent the average neuritelength (μm)±SEM.

FIG. 8(c). MAG(d1-3)-Fc Reverses Inhibition of Axonal Regeneration byWildtype MAG Expressed by CHO Cells. Cerebellar neurons (PND2) weregrown on MAG-expressing (MAG cells) or control CHO cells, in thepresence (+MAG1-3) or absence of MAG(d1-3)-Fc. Neurite length (μm) wasmeasured as described in FIG. 1. Results represent the average neuritelength (μm)±SEM.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be fully understood,the following detailed description is set forth.

The term "MAG derivative" refers to a molecule comprising at least oneMAG extracellular domain, wherein the MAG molecule has been altered(e.g., by recombinant DNA techniques to make chimera with portions ofother molecules fused to the MAG molecule, or by chemical or enzymaticmodification) or mutated (e.g., internal deletions, insertions,rearrangements and point mutations). MAG derivatives, unless otherwisenoted, retain MAG activity.

The terms "MAG bioactivity" and "MAG biological activity" refer to theability of a molecule, especially an altered or mutant form of MAG, toinhibit or promote neurite outgrowth of a selected neuronal cell type ofa particular age, as detected in a neurite outgrowth assay such as thosedescribed herein, in qualitatively the same direction as cell-surface orsoluble MAG.

The term "MAG binding activity" refers to the ability of a molecule,especially an altered or mutant form of MAG, to compete withcell-surface MAG or soluble MAG for sialic-acid dependent neuron bindingin an assay such as those described herein. For example, preferredinhibitors of MAG retain MAG binding activity but have reduced or absentMAG bioactivity.

The term "MAG activity" refers generically to MAG bioactivity andbinding activity as described above.

The term "modified derivative of sialic acid" refers to a sialic acidresidue that has been modified chemically or enzymatically, especiallyto add or exchange chemical groups or side chains onto reactivepositions of the molecule. Sialic acids are a family of nine-carbonacidic sugars which are derivatives of neuraminic acid and which areoften at the termini of cell-surface carbohydrates. "NeuAc" stands forN-acetylneuraminic acid; "GalNAc" stands for N-acetylgalactosamine.

MAG Is a Potent Inhibitor of Neurite Outgrowth

As described above, MAG had been shown previously to promote neuriteextension from particular types of neurons. In addition, MAG wasbelieved to be involved in the initiation of myelin formation. Thepresent invention demonstrates a novel role for MAG as an inhibitor ofaxonal outgrowth and hence of nerve regeneration in the central nervoussystem (CNS) and the peripheral nervous system (PNS).

Selected neuronal cell types may be isolated from animals at increasingtimes in postnatal days (PND) according to the procedures described inExample 1. Neurons representing a single cell type may be isolated andtested alone, or if desired, mixed populations of cells comprising oneor more neuronal cell types in the presence or absence of non-neuronalcells may also be tested.

The present invention provides an in vitro assay for determining whetherneurite outgrowth from a particular type of neuron at a particular ageis stimulated or inhibited in the presence of MAG. Isolated neurons ofchoice may be cultured on a monolayer comprising a growth-permissivesubstrate in the presence or absence of bound MAG, and comparativeneurite outgrowth may be measured. Preferably the growth-permissivesubstrate comprises mammalian fibroblast cells which have beenengineered to express MAG on their cell surfaces. MAG-expressing cellsmay be engineered using the procedures described in Example 2. Neuriteoutgrowth on MAG-expressing cells may then be compared to neuriteoutgrowth on control cells that do not express cell-surface MAG.

As shown in FIG. 1, MAG is a potent inhibitor of axonal growth fromcerebellar neurons from all ages of rats tested, newborn to adult. Thiswas determined by co-culturing the neurons on transfected Chinesehamster ovary (CHO) cells expressing MAG and comparing the length of theneurites extended to those extended on control transfected CHO cells notexpressing MAG (see Example 2). Four different MAG-expressing cell lineswere used and each had the same effect and inhibited neurite outgrowthby at least 70% compared to control cells at all ages tested (FIG. 1;see also Mukhopadhyay et al., Neuron, 13, pp. 757-67 (1994)). Thereversal of neurite growth inhibition by anti-MAG antibodies and thelack of effect of CHO cells expressing another myelin protein, Po, onneurite outgrowth demonstrates that this inhibition is specific to MAG(Mukhopadhyay et al., supra). However, as discussed above, it had beenreported previously that MAG promotes neurite outgrowth from newborn DRGneurons (Johnson et al., Neuron, 3, pp. 377-385 (1989)).

To clarify this apparent discrepancy, dorsal root ganglia (DRG) neuronsfrom one- to twenty-day old rats were tested in the neurite outgrowthassay on CHO cells as described in Example 2. As shown in FIG. 2, MAGenhanced neurite outgrowth of DRG neurons from two-day old rats;neurites were almost twice as long on MAG-expressing cells compared tocontrol cells. On the other hand, when DRG neurons from adult rats weretested, MAG inhibited neurite outgrowth by about 40%. A more detailedtime course of the effect of MAG on neurite outgrowth from DRG neuronsof different ages revealed that the transition from promotion toinhibition takes place at about post-natal day 4 (FIG. 2). Hence,depending on the age and the type of neuron, MAG can either promote orinhibit neurite outgrowth.

MAG inhibits axonal outgrowth from many types of neurons

Characterization of the effect of MAG on a variety of neuronalpopulations would aid in defining the requirements for enhancedregeneration after injury in various regions of the nervous system. Toestablish how other populations of neurons behave in response to MAG,various different neuronal cell types were isolated from one-day oldrats (PND1) as described in Example 1. Isolated retinal ganglion (RG),hippocampal (HN), motor neurons (MN) and superior-cervical ganglion(SCG) were tested in CHO cell neurite outgrowth assays as described inExample 2. As shown in FIG. 3, MAG is a potent inhibitor of neuritegrowth in all cell types tested. Thus, MAG likely plays an importantrole in the lack of neural regeneration in all areas of the nervoussystem tested to date.

These results show that the transfected mammalian cell assay describedin Example 2 is an effective assay whereby both the inhibition andpromotion of neurite outgrowth by MAG can be monitored andcharacterized. This assay can also be used to screen and identify agentsthat can block (or enhance) MAG bioactivity, thereby altering itsinhibition or stimulation of axonal outgrowth in the nervous system(Example 3). Such agents are called herein "MAG-dependent neurite growthaltering agents."

MAG inhibits axonal outgrowth by binding to a sialic acid-bearingglycoprotein on neurons

MAG binds to all types of neurons tested in a sialic acid-dependentfashion (Kelm et al., Curr. Biol., 4, pp. 965-72 (1994)). FIG. 4 showsthe results of an aqueous MAG-Fc neuron binding assay which wasperformed essentially as described in Kelm et al. This experimentconfirms that the binding of MAG to isolated PDN1 cerebellar neurons(whose outgrowth is inhibited by MAG) is abolished either by inclusionof anti-MAG monoclonal antibody 513 or by sialidase treatment of neuronsbefore the binding reactions. Sialidase is an enzyme which removessialic acid from glycoconjugates. Similarly, the binding of MAG toisolated PDN1 DRG neurons (whose outgrowth is promoted by MAG) isinhibited by inclusion of anti-MAG monoclonal antibody 513 and to aslesser extent, by sialidase treatment.

To determine if the sialic acid-dependent binding of MAG to neurons isthe event that signals inhibition or promotion of axonal growth andregeneration, neurite outgrowth assays such as those described inExample 2 were performed after the isolated neurons had been treatedwith sialidase (Example 7). The inhibition of axonal regeneration wasreversed by about 50% when PND2 cerebellar neurons were desialyated(FIG. 5). Similarly, when newborn (PND1) DRG neurons were desialyated,promotion of axonal outgrowth by MAG was completely abolished (FIG. 6).

Axonal outgrowth assays such as those described in Example 2 were alsoperformed in the presence of small, free sialic acid-bearing sugars.These sugars can compete with the sialic acid components of the neuronalsurface for MAG binding and thereby block the inhibition (TABLE 1) orpromotion (TABLE 2) of neurite growth by MAG. Inclusion of increasingconcentrations of either of the small sialic acid-bearing sugars2,3-dideoxy sialic acid (DD-NANA) or sialo 2,3-α lactose (SL) reversedthe inhibition of axonal growth by MAG by between 40-56% (TABLE 1) andabolished the promotion of neurite outgrowth by MAG completely (TABLE2).

                  TABLE 1    ______________________________________    SMALL SIALIC ACID-SUGARS BLOCK    THE INHIBITION OF AXONAL GROWTH BY MAG                Conc.               % Reversal of    Sugar       (mM)      Cell      Inhibition    ______________________________________    DD NANA      0        control    0    DD NANA     20        control    0    DD NANA      0        MAG        0    DD NANA      1        MAG       15%    DD NANA     10        MAG       45%    DD NANA     20        MAG       64%    SL           0        control    0    SL           9        control    0    SL           0        MAG        0    SL           1        MAG       11%    SL           5        MAG       42%    SL           9        MAG       26%    ______________________________________

Neurite outgrowth was compared for cerebellar neurons from PND 2animals, grown on MAG-expressing and control CHO cells as describe inExample 3.

                  TABLE 2    ______________________________________    SMALL SIALIC-ACID SUGARS BLOCK THE    PROMOTION OF AXONAL GROWTH BY MAG                 Conc.               % Reversal    Sugar        (mM)     Cell       of Promotion    ______________________________________    DD NANA       0       control     0    DD NANA      20       control     0    DD NANA       0       MAG         0    DD NANA       1       MAG         0    DD NANA      10       MAG        51%    DD NANA      20       MAG        97%    SL            0       control     0    SL           18       control     0    SL            0       MAG         0    SL            1       MAG         0    SL            5       MAG        96%    SL           18       MAG        98%    ______________________________________

Neurite outgrowth was compared for DRG neurons from PND 2 animals, grownon MAG-expressing and control CHO cells as describe in Example 3.

The experiments described above demonstrate that MAG biological activityis normally dependent on the ability of MAG to bind to a sialicacid-bearing component on the surface of neurons. It is thus envisionedthat a variety of agents that can disrupt the ability of MAG to bind tothis sialic acid-bearing component will function in vivo as MAGinhibitors and will thus be useful for regulating, and especially forpromoting neuronal growth in the CNS and PNS.

Inhibitors of MAG binding activity include but are not limited toanti-MAG antibodies, free sialic acid-bearing sugars, modifiedderivatives of sialic acid attached to a sugar, a sialic acid-bearingsugar attached to a protein or lipid carrier molecule, a modified sialicacid-bearing sugar attached to a protein or lipid carrier molecule andsialic acid glycopeptides or glycoproteins.

As shown above, inhibitors of MAG binding activity also include enzymesthat can alter or remove sialic acid residues, especially those having aNeu5Acα2→3Galβ1→3GalNAc (3-O) structure, which mediates MAG binding toneuronal surfaces in the PNS or CNS. Preferred compositions of thisembodiment comprise sialidase (a neuraminidase) and sialyl transferasesthat alter the structure and/or lower the effective concentration ofNeu5Acα2→3Galβ1→3GalNAc ("3-O") sialyated glycans.

Identifying MAG-dependent Growth Regulating Agents

Putative new MAG-dependent neurite growth regulating agents may betested using the procedures described in Example 3. A test agent isidentified as a MAG inhibitor when it promotes neurite growth from acell type inhibited by MAG or inhibits neurite growth from a cell typestimulated by MAG. Similarly, an agent is a MAG agonist when it promotesneurite growth from a cell type stimulated by MAG or inhibits neuritegrowth from a cell type inhibited by MAG.

Soluble MAG is a potent inhibitor of axonal regeneration

This invention provides a second method for assaying the effects of MAGon axonal growth and for identifying MAG-dependent neurite growthaltering agents. The method involves culturing separate samples of aselected neuronal cell type on a growth-permissive substrate lacking MAGin the presence of either a known concentration of a traceable, solubleform of MAG or of a control protein lacking MAG activity. The neuroncultures are then incubated with a known relative concentration of atest agent for a time sufficient to allow neurite growth, and the amountof neurite growth in the cells cultured in the presence or absence ofsoluble MAG compared (Example 5). An agent that changes the relativegrowth of neurites from cells cultured in the presence and absence ofsoluble MAG is identified as a MAG-dependent neurite growth alteringagent.

In a preferred embodiment, the growth-permissive substrate lacking MAGcomprises a monolayer of mammalian cells that do not expresscell-surface MAG, such as COS or NIH 3T3 cells. A wide variety ofmammalian cell lines, such as fibroblast and epithelial cells, may beused and are well known to those of ordinary skill in the art. Thepresent invention is not limited by the cell types which may be employedto make such growth-permissive monolayers that do not comprise boundMAG.

In another preferred embodiment, the growth-permissive substrate lackingMAG comprises an immobilized monolayer of a purified, growth-promotingfactor. It is well known in the art that neuronal cells may be culturedon growth-promoting monolayers comprising collagen or fibronectin. Apreferred neuronal growth-promoting factor according to the presentinvention which may be immobilized onto a monolayer is the L1glycoprotein.

In preferred embodiments of this method, the soluble form of MAG is aMAG-FC fusion protein, and the soluble control protein lacking MAGactivity is a MUC18-Fc fusion protein (Example 4). Preferred traceablefusion proteins are radioactively or fluorescently labeled usingcommercially available reagents and methods well known in the art.

FIG. 7a shows the results of an assay performed according to theprocedures described in Example 4. In this assay, neurons were grown ona substrate comprising the purified growth-promoting molecule termed"L1". MAG in a soluble form, consisting of the extracellular domain ofMAG fused to the Fc region of IgG (MAG-Fc) was added to the growingneurons (MAG-Fc; Example 4). As the concentration of MAG-Fc wasincreased, inhibition of neurite outgrowth increased, while a controlchimera, MUC18-Fc, at the same concentration had no effect (FIG. 7a).Furthermore, inhibition of axonal regeneration by MAG-Fc could bereversed by either adding a monoclonal antibody directed against MAG orby desialyating the isolated neurons prior to the assay (FIG. 7b).Soluble MAG-Fc can also inhibit axonal regeneration from cerebellarneurons grown on a monolayer of fibroblasts (FIG. 7c).

Importantly, using this assay, an inhibitor of MAG activity wasidentified which can bind to neurons without inhibiting axonalregeneration and can reverse the inhibitory effects of wildtype MAG: Atruncated form of MAG-Fc, consisting of the first three, rather than thenormal five Ig-like extracellular domains of MAG fused to an IgG Fcdomain ("MAG(d1-3)-Fc"; Example 4), specifically bound to cerebellarneurons (FIG. 8a). FIG. 8a shows that soluble Fc chimera consisting ofall five ("MAG(d1-5)") or the first three ("MAG(d1-3)") Ig-like domainsof MAG could bind to cerebellar neurons from PND2 rats in a reactionwhich was completely inhibited by the presence of anti-MAG monoclonalantibodies. A control Fc chimeric protein (MUC18) did not bind toneurons either in the presence or absence of anti-MAG antibodies.

However, when added to neurons growing on a monolayer substrate of L1,unlike the normal MAG-Fc chimera, MAG(d1-3)-Fc chimera had no effect onaxonal regeneration (FIG. 8b). It is thus likely that MAG(d1-3)-Fc cancompete with full-length MAG for binding to neurons because MAG(d1-3)-Fcat a concentration of 50 μg/ml can reverse by about 40% inhibition ofaxonal regeneration by full-length MAG expressed by CHO cells (FIG. 8c).

The above experiments demonstrate that altered and/or mutated forms ofsoluble MAG which harbor one or more mutations in the MAG molecule thatreduce or eliminate its ability to inhibit or promote neurite outgrowthbut do not significantly diminish the binding of the altered or mutantform of MAG to neuronal surfaces may be useful inhibitors of MAGactivity when administered in vivo (Example 8). The most preferredaltered/mutant forms of MAG of the present invention are solublemolecules comprising a truncated form of-MAG-Fc consisting of the firstthree of the five extracellular Ig-like domains of MAG fused to animmunoglobulin Fc domain ("MAG(d1-3)-Fc").

It is envisioned that other more specific mutations (especially pointmutations or small internal deletions) may be made to MAG Ig-likedomains that will also reduce or eliminate its ability to inhibit orpromote neurite outgrowth without significantly diminishing the bindingof the mutant form of MAG to neuronal surfaces. A mutational analysiswill likely lead to the identification of a localized "MAG neuritegrowth signaling site" necessary for activating the downstream cellularsignals that are involved in mediating neurite growth regulation. It isenvisioned that certain mutations targeted especially to the fourth andfifth domains which are deleted in the MAG(d1-3)-Fc chimeric protein,and/or to the junction between Ig-like domains three and four in MAGwill be useful in this regard.

Pharmaceutical Compositions and Treatments Using MAG Derivatives andInhibitors

The MAG-dependent neurite growth regulating agents of this invention maybe formulated into pharmaceutical compositions and administered in vivoat an effective dose to treat the particular clinical conditionaddressed. Administration of one or more of the pharmaceuticalcompositions according to this invention will be useful for regulatingand for promoting neural growth or regeneration in the nervous system,for treating injuries or damage to nervous tissue or neurons, and fortreating neural degeneration associated with traumas to the nervoussystem, disorders or diseases. Such traumas, diseases or disordersinclude, but are not limited to: aneurysms, strokes, Alzheimer'sdisease, Parkinson's disease, Creutzfeldt-Jacob disease, kuru,Huntington's disease, multiple system atrophy, amyotropic lateralsclerosis (Lou Gehrig's disease), and progressive supranuclear palsy.

Determination of a preferred pharmaceutical formulation and atherapeutically efficient dose regiment for a given application iswithin the skill of the art taking into consideration, for example, thecondition and weight of the patient, the extent of desired treatment andthe tolerance of the patient for the treatment.

Administration of the MAG derivatives and inhibitors of this invention,including isolated and purified forms, their salts or pharmaceuticallyacceptable derivatives thereof, may be accomplished using any of theconventionally accepted modes of administration of agents which are usedto treat neuronal injuries or disorders.

Soluble altered and mutated forms of MAG such as those described hereinare prepared from the culture media of transfected cells, e.g., COScells (fibroblasts), transfected with expression plasmids encoding thecDNAs for these forms of MAG (Example 4). The soluble MAG molecules,such as MAG-Fc, are secreted by these cells. It is anticipated that, ashas been carried out for hybridoma cells that secrete antibodies(Schnell, L. and Schwab, M. E., Nature, 343, pp. 269-72 (1990); Schnellet al., Nature, 367, pp. 170-73 (1993), COS cells or other transfectantssecreting the soluble MAG-Fc chimera may be implanted into damagedspinal cord. The cells will secrete MAG-inhibiting forms of altered ormutated MAG-Fc, which prevents the endogenous MAG from interacting withthe neuronal surface and thus prevents inhibition of axonal growth andregeneration by endogenous MAG.

About 2×10⁶ transfected cos cells will secrete about 1 mg of MAG-Fc overa 5-day period. A concentration of 50 μg/ml of mutated MAG-Fceffectively reverses the inhibitory effects of wildtype MAG. Finally,within the perineurium of an adult rat spinal cord is a volume of about0.5 ml. Therefore, if 2×10⁶ mutated or altered MAG-Fc-secreting COScells are implanted into an injured spinal cord, then the concentrationof MAG-Fc should be maintained at about 400 μg/ml, i.e., 8-fold moreconcentrated than the concentration shown herein to be effective incultured cells. Finally, calculations to correct for the differencebetween the volume of the perineurium of an adult rat spinal cordcompared to the subject being treated can be made by one of skill in theart. Transfected cells, secreting other "reversing" mutated forms of MAGor MAG "blocking" peptides can be administered to the site of neuronalinjury or degeneration in a similar manner.

Likewise, other MAG inhibitors and regulators of this invention, e.g.,sialidases and sialyltransferases, free, protein- or lipid-attachedsialic acid-bearing sugars, glycopeptides or glycoproteins, can also bedelivered by spinal implantation (e.g., into the cerebrospinal fluid) ofcells engineered to secrete MAG regulating agents according to thisinvention. Cell secretion rates of the agent are measured in cellculture and then extrapolated.

Optionally, transfected cells that secrete MAG regulating agents may beencapsulated into immunoisolatory capsules or chambers and implantedinto the brain or spinal cord region using available methods that areknown to those of skill in the art. See, e.g., WO 89/04655; WO 92/19195;WO 93/00127; EP 127, 989; U.S. Pat. Nos. U.S. Pat No. 4,298,002; U.S.Pat. No. 4,670,014; U.S. Pat. No. 5,487,739 and references citedtherein, all of which are incorporated herein by reference.

For MAG regulating agents that can not be secreted by transfected cells,a pump and catheter-like device may be implanted at the site of injuryto administer the agent on a timely basis and at the desiredconcentration, which can be selected and empirically modified by one ofskill in the art. Such pharmaceutical delivery systems are known tothose of skill in the art. See, e.g., U.S. Pat. No. 4,578,057 andreferences cited therein, which are incorporated herein by reference.

The pharmaceutical compositions of this invention may be in a variety offorms, which may be selected according to the preferred modes ofadministration. These include, for example, solid, semi-solid and liquiddosage forms such as tablets, pills, powders, liquid solutions orsuspensions, suppositories, and injectable and infusible solutions. Thepreferred form depends on the intended mode of administration andtherapeutic application. Modes of administration may include oral,parenteral, subcutaneous, intravenous, intralesional or topicaladministration.

The MAG derivatives and inhibitors of this invention may, for example,be placed into sterile, isotonic formulations with or without cofactorswhich stimulate uptake or stability. The formulation is preferablyliquid, or may be lyophilized powder. For example, the MAG derivativesand inhibitors may be diluted with a formulation buffer comprising 5.0mg/ml citric acid monohydrate, 2.7 mg/ml trisodium citrate, 41 mg/mlmannitol, 1 mg/ml glycine and 1 mg/ml polysorbate 20. This solution canbe lyophilized, stored under refrigeration and reconstituted prior toadministration with sterile Water-For-Injection (USP).

The compositions also will preferably include conventionalpharmaceutically acceptable carriers well known in the art (see forexample Remington's Pharmaceutical Sciences, 16th Edition, 1980, MacPublishing Company). Such pharmaceutically acceptable carriers mayinclude other medicinal agents, carriers, genetic carriers, adjuvants,excipients, etc., such as human serum albumin or plasma preparations.The compositions are preferably in the form of a unit dose and willusually be administered one or more times a day.

The pharmaceutical compositions of this invention may also beadministered using microspheres, liposomes, other microparticulatedelivery systems or sustained release formulations placed in, near, orotherwise in communication with affected tissues or the bloodstream.Suitable examples of sustained release carriers include semipermeablepolymer matrices in the form of shaped articles such as suppositories ormicrocapsules. Implantable or microcapsular sustained release matricesinclude polylactides (U.S. Pat. No. 3,773,319; EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22, pp. 547-56 (1985)); poly(2-hydroxyethyl-methacrylate) or ethylenevinyl acetate (Langer et al., J. Biomed. Mater. Res., 15, pp. 167-277(1981); Langer, Chem. Tech., 12, pp. 98-105 (1982)).

Liposomes containing MAG derivatives and inhibitors can be prepared bywell-known methods (See, e.g. DE 3,218,121; Epstein et al., Proc. Natl.Acad. Sci. U.S.A., 82, pp. 3688-92 (1985); Hwang et al., Proc. Natl.Acad. Sci. U.S.A., 77, pp. 4030-34 (1980); U.S. Pat. Nos. 4,485,045 and4,544,545). Ordinarily the liposomes are of the small (about 200-800Angstroms) unilamellar type in which the lipid content is greater thanabout 30 mol. % cholesterol. The proportion of cholesterol is selectedto control the optimal rate of MAG derivative and inhibitor release.

The MAG derivatives and inhibitors of this invention may also beattached to liposomes, which may optionally contain other agents to aidin targeting or administration of the. compositions to the desiredtreatment site. Attachment of MAG derivatives and inhibitors toliposomes may be accomplished by any known cross-linking agent such asheterobifunctional cross-linking agents that have been widely used tocouple toxins or chemotherapeutic agents to antibodies for targeteddelivery. Conjugation to liposomes can also be accomplished using thecarbohydrate-directed cross-linking reagent 4-(4-maleimidophenyl)butyric acid hydrazide (MPBH) (Duzgunes et al., J. Cell. Biochem. Abst.Suppl. 16E 77 (1992)).

Utility of MAG Derivatives and Inhibitors

The discovery that MAG is a potent inhibitor of axonal regeneration haspotential clinical use in the situations of nervous system injury--bothof the peripheral and central nervous systems--and in particular for CNSinjury. The mammalian central nervous system does not regenerate afterinjury even though there are many molecules present that promote andencourage a nerve to grow. The result is paralysis or brain damage. Ithas been shown that there are molecules present in the adult CNS thatwill actively prevent a nerve from regenerating. It is anticipated thatif these inhibitory molecules can be first identified and subsequentlyblocked, then an environment permissive for regeneration could beengineered.

The first step is to identify what the inhibitory molecules are. MAG isthe first such molecule to be identified in myelin. Using the assaysystems established herein to monitor the inhibitory effects of MAG,strategies can now be designed with MAG as a target such that itsinhibitory function is blocked. Such an agent can then be administeredto damaged nerves reversing the inhibitory effects of MAG in vivo andallowing nerve regeneration to proceed.

The assays of the present invention are useful for identifying agentslikely to reverse inhibition of nerve regeneration by MAG. Using theassay systems described herein, the inhibitory effects of MAG were shownto be blocked/prevented from functioning by agents such as sialidases orsmall sialic acid-bearing sugars and by soluble, mutated forms of MAG.These agents, or modified forms of these agents that can either increaseor decrease their affinity for MAG or for its receptor, may beadministered to damaged nerves, reversing the inhibitory effects of MAGin vivo and allowing regeneration to proceed.

In addition, the properties of MAG as a negative guidance cue can beused to guide regenerating axons to their correct target and keep themon the correct path. For this purpose, MAG, or different domains of MAG,can be administered to the precise regions of the regenerating nervoustissue to contain growth along exact pathways.

As shown herein, MAG binds to a sialic acid-bearing glycoprotein onneurons to bring about inhibition of nerve growth and regeneration in awide variety of neuronal cell types. And also as shown herein, when thesialic acid residues of neurons are removed with enzymes termedsialidases, these inhibitory effects of MAG are reversed. Similarly,small sialic acid-bearing sugars and derivatives thereof can bind toMAG, prevent it from interacting with the sialic acid glycoprotein onneurons and prevent its inhibition of axonal regeneration. It isanticipated that in vivo, after injury, application of MAG inhibitorssuch as sialidases, free-small sialic-bearing sugars or modifications ofsialic acid attached to other sugars, small sialic acid-bearing sugarscovalently attached to protein carrier molecules or lipids or smallsialic acid glycoproteins or glycopeptides, either individually or invarious combinations, will block the inhibitory effects of MAG and/orother inhibitory molecules that act through a sialic acid-bearingreceptor, and encourage axonal regeneration to take place. Similarly,small peptides or peptide fragments of MAG , mutated and altered formsof MAG and antibodies to MAG may block the interaction of endogenous MAGwith neurons and allow nerve regeneration.

As shown herein, a mutated, soluble form of MAG can bind to neurons, butitself does not inhibit axonal growth. Importantly, this mutated form ofMAG can reverse the inhibitory effects of wildtype MAG.

Finally, it is envisioned that MAG, MAG derivatives and MAG inhibitorsmay be used as a guidance cue in precise regions of the regeneratingnervous system to keep growing axons on the correct path and movingtowards the correct target.

All references cited herein are hereby incorporated by reference.

The following are examples which illustrate the methods of thisinvention used to identify the MAG-dependent neurite growth alteringagents, compositions of this invention which comprise such agents, andmethods comprising the administration of those compositions. Theseexamples should not be construed as limiting: the examples are includedfor the purposes of illustration only and the present invention islimited only by the claims.

EXAMPLE 1 Isolation of Different Neuronal Cell Types

Neurons were isolated essentially as described in Doherty et al.,Nature, 343, pp. 464-66 (1990); Neuron, 5, pp. 209-19 (1990); andKleitman et al., Culturing Nerve Cells, pp. 337-78, MIT Press,Cambridge, Mass./London, England (G. Banker and K. Goslin, Eds.) (1991).Briefly, for animals up to nine days of age, the cerebellum, retina,hippocampus, and spinal cord were removed from two animals. Like tissuewas combined and placed in 5 ml of 0.025% trypsin in PBS, triturated,and incubated for a further 10 minutes (min.) at 37° C. Trypsinizationwas stopped by addition of 5 ml DMEM containing 10% fetal calf serum(FCS) and cells were centrifuged at 800 rpm for 6 min. The cells wereresuspended to a single cell suspension in 2 ml of SATO containing 2%FCS. For DRG and SCG neurons, ganglia were removed from two animals andincubated in 5 ml of L15 medium containing 0.025% trypsin and 0.3%collagenase type I (Worthington) for 30 min. at 37° C. The ganglia weretriturated with a fire-polished Pasteur pipette. Trypsinization wasstopped by adding 5 ml of DMEM containing 10% FCS, centrifuged at 800rpm for 6 min., and resuspended in 2 ml of SATO containing 2% FCS. Cellswere counted with a Coulter counter.

EXAMPLE 2 Neurite Outgrowth Assays on Transfected CHO cells

Expression of MAG by Transfected CHO Cells

Chinese hamster ovary (CHO) cells deficient in the dihydrofolatereductase (dhfr) gene (Urlaub and Chasin, Proc. Natl. Acad, Sci. USA,77, pp. 4216-20 (1980)) were transfected with a MAG-cDNA expressionplasmid with the dhfr gene and the L-MAG cDNA in either a 5'-3' or, as acontrol, a 3'-5' orientation, cells with multiple copies of dhfr wereselected by growing in increasing concentrations of methotrexate, andthe expression of MAG by individual transfected CHO cell linescharacterized as described in Mukhopadhyay et al., Neuron, 13, pp.757-67 (1994), which is incorporated herein by reference. Transfectedcells were maintained in DMEM supplemented with 10% dialyzed FCS,proline (40 mg/liter), thymidine (0.73 mg/liter), and glycine (7.5mg/liter) at 37° C. in 5% CO₂.

The MAG-expressing transfected CHO cell line ("CHO-MAG2") described asMAG2 in that publication was deposited on Jun. 27, 1996 with theAmerican Type Culture Collection (ATCC) (Rockville, Md.) according tothe provisions of the Budapest Treaty, and was assigned the ATCCaccession number designated: CRL-12145!. All restrictions on theavailability to the public of the above ATCC deposit will be irrevocablyremoved upon the granting of a patent on this application.

Neurite Outgrowth Assays

Confluent monolayers of control and MAG-expressing CHO cells wereestablished over a 24-hour (h) period in individual chambers of an8-well tissue culture slide (Lab-Tek). Co-cultures were established asdescribed previously (Doherty et al., Nature, 343, pp. 464-66 (1990);Neuron, 5, pp. 209-19 (1990); Mukhopadhyay et al., Neuron, 13, pp.757-67 (1994)) by adding approximately 5000 cerebellar, dorsal rootganglion (DRG) and superior-cervical ganglion (SCG) neurons and 10,000retinal, hippocampal and spinal cord cells to the CHO monolayers.Culture medium was SATO containing 2% FCS. Where indicated, 20 mU of VCSwas included throughout the coculture period (see Example 4), ormonolayers were incubated with small oligosaccharides for one hourbefore adding the neuronal cell suspension and included throughout thecoculture period. After periods of time as indicated, the cocultureswere fixed for 30 min with 4% paraformaldehyde and permeabilized withice-cold methanol for 2 min. The cells were then blocked for 30 min withDMEM containing 10% FCS and incubated for 2 h with a rabbit polyclonalantibody against the neuronal marker GAP43 (1:4000). Cells were washedthree times with PBS-BSA (2%) and then incubated for 30 min at roomtemperature with a biotinylated donkey anti-rabbit Ig (1:300, Amersham),washed three times, and incubated with streptavidin-conjugated Texas Red(1:300, Amersham) for 30 min. After three more washes, the slides weremounted in Permfluor (Baxter) and viewed with a Zeiss fluorescentmicroscope. The length of the longest neurite for each GAP43-positiveneuron was determined using the Biological Detection System imageanalysis program (Pittsburgh).

Alternatively, other neuron-specific antibodies such asanti-neurofilament monoclonal antibodies, which are commerciallyavailable (e.g., Boehringer Mannheim, Sigma Immunochemicals), may beused starting at dilutions recommended by the manufacturer. Theappropriate species-specific, biotinylated anti-Ig secondary antibody isthen selected according to the species in which the primary anti-neuralantibody was generated. In addition, various vital dyes (e.g., MolecularProbes, Oregon) which stain neurites may be used in this assay in placeof a fluorescent neural-specific antibody.

EXAMPLE 3

Neurite Growth Assays with CHO Cells to Test Putative MAG-dependentNeurite Growth Altering Agents

The transfected CHO cell assay described in Example 2 may also be usedto screen and identify agents that alter neurite growth properties of aparticular neuronal cell type and age in a MAG-dependent fashion.Neurite outgrowth was compared for cerebellar (TABLE 1) and DRG (TABLE2) neurons from PND 2 animals, grown on MAG-expressing and control CHOcells as described in Example 2. Where indicated, small sialicacid-bearing sugars were included in the co-cultures at increasingconcentrations. 100% inhibition was taken as the difference in length ofneurites on control and MAG-expressing CHO cells. Results are the meanof at least two experiments, with at least 150 neurons measured for eachexperiment. DD-NANA=2,3-dideoxy sialic acid; SL=sialo 2,3-α lactose.

This assay may be used to test other putative MAG-dependent neuritegrowth regulating agents by including them in the coculture andmeasuring their effect in the presence and absence of cell-surface MAGas described above for the small sialic acid-bearing sugars.

EXAMPLE 4 Binding of Soluble MAG-Fc Chimeras to Neurons

Production of Immunoglobulin Fc-chimeric proteins

Expression plasmids encoding various forms of MAG-Fc (such as thosereferred to herein as MAG d1-5!-Fc), MAG d1-3!-Fc and a controlFc-chimeric protein MUC 18-Fc) were prepared as described in Kelm etal., Current Biol., 4, pp. 965-72 (1994) and references cited therein.For discussions and a general protocol for making soluble recombinantadhesin molecules, see D. L. Simmons, "Cloning cell surface molecules bytransient expression in mammalian cells," in Cellular Interactions inDevelopment--A Practical Approach, pp. 118-125, IRL Press, Oxford (Ed.D. A. Hartley) (1993); Development (Supp.), pp. 193-203 (1993); and P.R. Crocker and S. Kelm, "Methods for studying the cellular bindingproperties of lectin-like receptors," in Handbook of ExperimentalImmunology, pp. 1-30 (1995), which are incorporated herein by reference.

E. coli cell samples transformed with plasmids that express the MAGd1-5!-Fc, MAG d1-3!-Fc and MUC18-Fc chimeric proteins described in Kelmet al., supra, were deposited on Jun. 27, 1996 with the American TypeCulture Collection (ATCC) (Rockville, Md.) according to the provisionsof the Budapest Treaty, and were assigned the ATCC accession numbersdesignated as shown below:

    ______________________________________                    ATCC           CELL LINE                    Accession No.    ______________________________________    a)       MAG 1-3!-Fc                        98089    b)       MAG 1-5!-Fc                        98090    c)       MUC18-Fc   98088    ______________________________________

All restrictions on the availability to the public of the above ATCCdeposits will be irrevocably removed upon the granting of a patent onthis application.

Binding of Fc-Chimeras to Neurons

Plasmids encoding MAG-Fc, MAG d1-3!-Fc and MUC 18-Fc were transfectedinto COS cells and the Fc-chimeric proteins purified from the media asdescribed in Kelm et al., Current Biol., 4, pp. 965-72 (1994) and P. R.Crocker and S. Kelm, "Methods for studying the cellular bindingproperties of lectin-like receptors," in Handbook of ExperimentalImmunology, pp. 1-30 (1995).

Neuron binding assays were performed essentially as described inDeBellard et al., Mol. Cell. Neuroscience, 7, pp. 89-101 (1996), whichis incorporated herein by reference. Fc-chimeric proteins were adsorbedfor 3 h at 37° C. to wells of microtiter plates that had been coated for2 h at 37° C. with anti-human IgG at 15 μg/ml in 0.1M bicarbonatebuffer, pH 9.6. Prior to the binding assay, neurons were vitally labeledwith the fluorescent dye calcein AM (Molecular Probes) by incubating2×10⁶ neurons in 5 ml of 10 μM calcein AM in PBS for 15 min at 37° C.before being washed and resuspended in PBS. Where indicated, amonoclonal antibody directed against MAG (Boehringer Mannheim) wasincluded in the assay at a concentration of 10 μg/ml, and whereindicated, neurons were desialyated before being used. One hundred μl ofa suspension of vitally labeled neurons, containing 1-2×10⁵ cells wasadded to each well and allowed to incubate for 1 h at room temperature.The plates were washed three times with PBS applied to each well undergravity and the fluorescence was measured in a FluorImager (MolecularDynamics).

EXAMPLE 5 Neurite outgrowth Assays on a Growth Permissive Substrate inthe Presence or Absence of MAG-Fc Chimera

Growth Permissive Substrate comprising an L1-Fc Chimera

The L1 glycoprotein is a cell adhesion molecule (CAM) expressed on thesurface of a wide variety of mammalian neuronal cell types whichstimulates neurite outgrowth. Soluble L1-Fc chimera may be constructedusing procedures known to those of skill in the art (such as those citedin Example 3; Doherty et al., Neuron, pp. 57-66 (1995), incorporatedherein by reference). Soluble L1-Fc chimera, when presented to neurons,are as effective at promoting neurite outgrowth as the normal cellsurface-associated L1 (Doherty et al., supra, and references citedtherein which are incorporated herein by reference). As described inDoherty et al., L1-Fc chimera can stably associate with the surface offibroblast 3T3 cells or polylysine/collagen orpolylysine/fibronectin-coated substrates.

Individual wells of an eight-chamber tissue culture plastic slide(Lab-Tek, Nuc. Inc.) were incubated with 0.3 ml of 16.6 μg/mlpoly-1-lysine in sterile water for at least one hour under sterileconditions. Each well was washed twice with 400 μl of a 0.1M sodiumbicarbonate solution, pH 9.6, and then received 0.3 ml of 0.1M sodiumbicarbonate solution, pH 9.6, containing 15 μg/ml goat anti-human IgG(Fc-specific) monoclonal antibody (Sigma). The wells were incubated for2 h at 37° C., and washed three times with 0.4 ml of ice-cold DMEM. Eachwell then received 0.3 ml of DMEM containing 40 μg/ml of L1-Fc and wasincubated for 2-4 hours at 37° C. The wells were washed twice DMEM.

Neurite Outgrowth on L1-Fc Substrate

A Soluble MAG-Fc Binding Assay

Cerebellar neurons (post-natal days 2-7) were dissociated bytrypsinization as described in Example 1, except that the dissociatedneurons were resuspended in 5 ml of SATO medium containing 2% dialyzedFBS. To an individual well coated with a monolayer of L1-Fc as describedabove, 5.0×10⁴ cerebellar neurons were added, followed by either asingle concentration (about 50 μg/ml) or increasing concentrations(e.g., 0-30 μg/ml) of MAG-Fc or MUC18-Fc chimeric soluble proteins,depending on the experiment. Neurons were cultured overnight (about 16h) at 37°0 C., and then fixed and stained essentially as described inExample 2.

EXAMPLE 6 Neurite Outgrowth Assays Using Soluble MAG To IdentifyMAG-Dependent Neurite Growth Altering Agents

The neurite outgrowth binding assay using soluble MAG-Fc described inExample 5 may also be used to perform competitive neuron binding/growthexperiments to screen and identify new agents that alter the neuritegrowth properties of a particular neuronal cell type and age in aMAG-dependent fashion. One or more concentrations of the test agent wereincluded in the cocultures of Example 5, and the effect of the testagent in the presence and absence of soluble MAG assessed.

EXAMPLE 7 Neurite Outgrowth Assays With Desialyated Neurons

Single cell suspensions of different neurons at various postnatal ageswere washed and resuspended in phosphate-buffered saline (PBS).Approximately 2×10⁶ cells were incubated with 50 mU of Vibrio cholerasialidase (VCS, Calbiochem) (a neuraminidase) in a final volume of 0.5ml for 2 hours at 37° C. Neurons were washed with PBS and resuspended inSATO medium containing 2% FCS for neurite outgrowth experiments, or inPBS for neurite binding assays.

This procedure may be modified by using enzymes other than sialidasethat digest or otherwise modify carbohydrate structures (see, e.g., Kelmet al., Carbohydr. Res., 149, pp. 59-64 (1986), which is incorporated byreference herein). For example, sialyl transferases may be employed toalter or remove sialyated glycans on neuronal surfaces comprising sialicacid residues having a Neu5Acα2→3Galβ1→3GalNAc (3-O) structure to whichMAG binds.

EXAMPLE 8 In Vivo Delivery of MAG-Dependent Neurite Growth AlteringAgents

COS cells transfected with an expression plasmid that encodesMAG(d1-3)-Fc were cultured and the cultures assayed for the rate ofMAG(d1-3)-Fc secretion. Approximately 2×10⁶ cells--which secrete about 1mg of MAG(d1-3)-Fc over a 5-day period--are surgically implanted intothe cerebrospinal fluid surrounding the spinal cord of an injuredsubject in the vicinity of nerve damage in need of repair. Optionally,repeated administrations are performed. The cells secrete MAG(d1-3)-Fc,which is capable of inhibiting endogenous MAG activity in the myelin ofthe implant site, and neural regeneration is stimulated.

What is claimed is:
 1. A composition which comprises a pharmaceuticallyacceptable carrier and at least one MAG inhibitor in an amount effectivefor altering neural growth or regeneration in the nervous system,wherein the MAG inhibitor is a soluble chimeric protein which comprisesextracellular Ig-like domains one to three of MAG.
 2. The compositionaccording to claim 1, wherein the MAG inhibitor is MAG(d1-3)-Fc.
 3. Acomposition which comprises a pharmaceutically acceptable carrier and atleast one MAG inhibitor in an amount effective for altering neuralgrowth or regeneration in the nervous system, wherein the MAG inhibitoris a soluble chimeric protein which comprises extracellular Ig-likedomains one to five of MAG.
 4. The composition according to claim 3,wherein the MAG inhibitor is MAG(d1-5)-Fc.
 5. The composition accordingto any one of claims 1-4, wherein the MAG inhibitor can inhibit thebinding of endogenous MAG to neurons in the CNS or PNS.
 6. A method forregulating neural growth or regeneration in the nervous system whichcomprises the step of administering, in a manner which can affect thenervous system, a composition according to any one of claims 1-4.
 7. Themethod according to claim 6, wherein neural growth or regeneration ispromoted.
 8. A method for treating injuries or damage to nervous tissueor neurons which comprises the step of administering, in a manner whichcan affect the nervous system, a composition according to any one ofclaims 1-4.
 9. A method for treating neural degeneration associated witha disorder or disease which comprises the step of administering, in amanner which can affect the nervous system, a composition according toany one of claims 1-4.
 10. A soluble chimeric protein which comprisesextracellular Ig-like domains one to three of MAG.
 11. The MAG inhibitorMAG(d1-3)-Fc.
 12. A soluble chimeric protein which comprisesextracellular Ig-like domains one to five of MAG.
 13. The MAG inhibitorMAG(d1-5)-Fc.